A Novel Two-Dimensional Liquid Chromatography Combined with Ultraviolet Detection Method for Quantitative Determination of Pyridoxal 5′-Phosphate, 4-Pyridoxine Acid and Pyridoxal in Animal Plasma

Simple Summary Vitamin B6 is directly or indirectly involved in many key biological metabolic processes in the body in the form of coenzyme factors, and it is able to maintain the normal progress of biological responses at very low levels, playing an important role in animal health and disease. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. Its advantage of large volume injection provides adequate sensitivity. At the same time, multi-column is used to improve the shape of the peak, and the anti-interference ability is obviously improved. The system achieved rapid detection and stability quantification of PLP, PA, and PL. This method has been successfully applied to the plasma matrix of pigs, mice, and rats, providing a brand-new method for the determination of PLP, PA, and PL. Abstract Vitamin B6 is an indispensable micronutrient in organisms and is widely distributed in blood, tissues, and organs. Changes in the content and ratio of vitamin B6 can affect the entire physiological condition of the body, so it becomes particularly important to reveal the relationship between changes in its content and disease by monitoring vitamin B6 levels in the organism. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. First, PLP, PA, and PL were extracted with plasma: 0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and then derivatized. Enrichment and preliminary separation were performed on a one-dimensional column and automatically entered into a two-dimensional column for further separation. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves were >0.99. The detection limits for PLP, PA, and PL were 0.1, 0.2, and 4 nmol/L, respectively. The results showed that the system has high loading capacity, excellent resolution, and a good peak shape. This method is expected to provide applicability for the determination of PLP, PA, and PL in pharmacological, pharmaceutical, and clinical research.


Introduction
Vitamin B 6 participates in more than 100 enzymatic reactions as a coenzyme, including decarboxylation, racemization, transamination, elimination, and aldol cleavage, which

Instruments and 2D-LC Conditions
The equipment and chromatographic conditions were tested by a Shimadzu and ANAX combined system. The FLC-2420 column oven of the 2D-LC system was from ANAX (Changsha, China). Three high-pressure pumps (Pump A is a high-pressure gradient chromatography LC-20AT pump; Pump B and Pump C are high-pressure LC-20AT pumps), an SPD-20A UV detector, a SIL-20A auto-sampler (with a 600-µL injection loop), a CBM-20A system controller, and three six-port two-position FCV-12AH switching valves of the 2D-LC system are all from Shimadzu (Kyoto, Japan).
The first-dimensional liquid chromatogram (1D) LC separation used an SPY column (50 mm × 4.6 mm, 5 µm, Changsha, China; LC1). The mobile phase used for Pump C was methanol and 0.02 mol/L ammonium acetate (pH = 6.1) (5:95, v/v), with a flow rate of 1.0 mL/min. The second-dimensional (2D) LC separation used a C18 column (Waters Summetry ® , 4.6 × 150 mm, 5 µm; LC2). The mobile phase used for Pump A was methanol, acetonitrile, and 0.02 mol/L ammonium acetate (pH = 6.1) (9:6:85, v/v/v), with a flow rate of 1.0 mL/min. One-quarter of 0.02 mol/L ammonium dihydrogen phosphate (20 mM) was added to ammonium acetate to improve the chromatographic peak pattern. The chromatographic separations were performed in the isometric elution. The UV response of PLP and PL after derivatization was enhanced, and the maximum UV absorption wavelength was changed. Therefore, it was feasible to change the absorption wavelength of PLP and PL through the derivatization reaction to increase the response value to improve the sensitivity. For general considerations, it was determined that 310 nm was the UV detection wavelength of the three vitamin B 6 samples. The separation process was set up at 40 • C. PLP, PA, and PL were enriched online on a one-dimensional column separated in the first dimension and then entered into a second-dimensional column for further separation and detection. The two-dimensional systems were connected via an Intelligent Fluid Path Control System (TRS) using a "center cut" to transfer the target. The transfer time procedures for PLP, PA, and PL are shown in Table 1.

Standard Solutions, Calibration Curves and Quality Control Samples
Standard stock solutions of PLP and PL were dissolved in a small volume of 1 mol/L HCl and diluted to 100 mmol/L in ultrapure water, respectively. PA standard stock solution was dissolved in 1 mol/L NaOH and diluted to 10 mmol/L in ultrapure water. Stock solutions were transferred to brown bottles and frozen at −80 • C.
The calibration curves were obtained by plotting the peak areas of PLP, PA, and PL against their corresponding concentrations. All samples, including the calibrated samples and quality control samples (QCs) at all levels, were divided into aliquots and placed in Eppendorf tubes (2.0 mL), stored at −80 • C, and brought to room temperature before use.

Sample Pretreatment
Blood was collected from the hearts of rats and mice after anesthesia. The blood was placed in anticoagulant tubes and centrifuged at 4 • C at 3000 rpm for 15 min immediately. The supernatant plasma was separated in brown centrifuge tubes. The collected porcine anterior venous blood was placed in an anticoagulant tube and centrifuged at 3000 rpm at 4 • C for 15 min immediately, and the supernatant plasma was collected in a brown centrifuge tube, all of which was frozen at −80 • C.

The Extraction Procedure
Sixty microliters of fresh plasma from pigs, mice, and rats were transferred into a 2-mL brown centrifuge tube; 120 µL of 0.6 M trichloroacetic acid solution was added, vortexed for 30 s, diluted with 180 µL of ultrapure water, mixed well, allowed to stand at 50 • C for 5 min, and centrifuged at 10,000 rpm at 4 • C for 10 min; and the supernatant was collected.

The Derived Procedure
Three hundred microliters of mixed standard solution or blank plasma supernatant was transferred into a 1.5-mL brown centrifuge tube, mixed well with 350 µL semicarbazide solution at 10 mmol/L, reacted in a constant temperature shaker at 50 • C for 20 min at 300 rpm in the dark, passed through a 0.22-µm filter membrane, and tested by a wellbalanced 2D-LC system.
2.6. 2D-LC Capability Assessment 2.6.1. 2D-LC Online Processing Capability Assessment Online processing capability assessment of 2D-LC was performed experimentally to avoid shifts and distortions of the chromatographic peaks due to increasing injected volumes. Using a mixed standard solution containing 10 µmol/L, after pretreatment, 10,  20,50,100,200, and 500 µL were injected according to the chromatographic conditions, and the peak area (As) values of PLP, PA, and PL were used as the dependent variable (Y). The sample quantity (Am) was the independent variable (X), and the regression curve was fitted by the least squares method to obtain a fitting equation.

2D-LC Transfer Capability Assessment
To realize the transfer of PLP, PA, and PL from the one-dimensional column to the two-dimensional column, the degree of transfer of the three targets is a key indicator for assessing the transfer performance of the 2D-LC system; that is, the transfer recovery and transfer precision are calculated by the PLP, PA, and PL peak areas under 2D-LC conditions and LC2 conditions, and the transfer recovery (Rtr) is expressed by the ratio of the two; the transfer precision is expressed by the coefficient of variation (CV%) among the PLP, PA, and PL peak areas obtained multiple times under 2D-LC conditions.
After pretreatment of the mixed standard solutions of PLP, PA, and PL with different concentrations, the samples were injected under LC2 and 2D-LC conditions. Each concentration was repeated three times, and the transfer recovery (Rtr) and transfer precision (CV%) of PLP, PA, and PL were calculated.

Method Validation Procedure
The method validation was performed in accordance with the U.S. Bioanalytical Method Validation Guidelines [38]. In the process of analysis and verification, we tested the selectivity, sensitivity (limit of detection (LOD) and limit of quantification (LOQ)), linearity, precision (intra-and interassay variation), accuracy, recovery, and stability of PLP, PA, and PL in vitamin B 6 compounds.
Six blank samples were selected from plasma biological samples of pigs, mice, and rats for analysis. Compared with the injection analysis of the alternative matrix (Milli-Q water), their interference in the retention time and selectivity of PLP, PA, and PL were evaluated.
LOD and LOQ are defined as the lowest concentrations in the matrix that can be detected and quantified. Their signal-to-noise ratios (S/N) were ≥3.3 and 10, respectively. A standard curve was established using LOQ as the starting concentration point, the injected concentration (C) as the independent variable (X), and the peak area (As) as the dependent variable (Y), and the regression curve was fitted according to the least squares method with R 2 > 0.99 to obtain the fitting equations for PLP, PA, and PL.
The precision and accuracy were determined by analyzing QC solutions. Plasma LLOQ, LQC, MQC, and HQC solutions of pigs, mice, and rats were pretreated and injected according to 2D-LC conditions. Each concentration sample was measured five times for three consecutive days, and intra-day and inter-day precision, accuracy, and recovery rates of PLP, PA, and PL were calculated.
Pig, mouse, and rat plasma QCL, QCM, and QCH were stored in a 4 • C for three days and six replicates of each concentration; after pretreatment, samples were injected according to 2D-LC chromatographic conditions. Short-term stability of QCs in 4 • C was verified.

Application of the Method
In this experiment, the calibration curves of pig, mouse, and rat plasma samples were established for simultaneous quantitative analysis of PLP, PA, and PL, and the method was studied to prove the applicability of the method.

Extraction Condition Results
The preparation of vitamin B6 sample requires extraction and purification steps to reduce the pollution by impurities of the equipment and to ensure the precision and stability of the analysis method. Under the same pretreatment conditions, the recovery of trichloroacetic acid and organic reagent-treated samples was investigated. As shown  Table 2; the extraction yields of PLP were generally low; and the extraction recoveries of PLP, PA, and PA were relatively good at methanol: acetonitrile = 6:4 (v/v). Trichloroacetic acid (0.6 M) was selected as a precipitating protein reagent to further optimize the extraction conditions of vitamin B 6 .

Extraction Condition Results
The preparation of vitamin B6 sample requires extraction and purification steps to reduce the pollution by impurities of the equipment and to ensure the precision and stability of the analysis method. Under the same pretreatment conditions, the recovery of trichloroacetic acid and organic reagent-treated samples was investigated. As shown in Figure 1, 0.6 M TCA had good recovery; the extraction recoveries of different organic reagents are shown in Table 2; the extraction yields of PLP were generally low; and the extraction recoveries of PLP, PA, and PA were relatively good at methanol: acetonitrile = 6:4 (v/v). Trichloroacetic acid (0.6 M) was selected as a precipitating protein reagent to further optimize the extraction conditions of vitamin B6.  Note: "Met" means methanol; "Ace" means acetonitrile; "W" means water.
Under the same conditions, 50 μL of mouse plasma were used to investigate the addition volume of TCA and the dilution ratio of ultrapure water, as shown in Table 3. Plasma was extracted with more than two volumes of 0.6 M TCA, and the recoveries of PLP, PA, and PL were good; the plasma was diluted without water, or the dilution volume was less than 50 μL (plasma: 0.6 M TCA: water = 1:2:1, v/v/v); the recoveries of PLP and PL were low; the recoveries of PLP were higher than 120% at the dilution volume of 100 μL; the recoveries of PL were within the normal range, and the PA chromatographic peak  Note: "Met" means methanol; "Ace" means acetonitrile; "W" means water.
Under the same conditions, 50 µL of mouse plasma were used to investigate the addition volume of TCA and the dilution ratio of ultrapure water, as shown in Table 3. Plasma was extracted with more than two volumes of 0.6 M TCA, and the recoveries of PLP, PA, and PL were good; the plasma was diluted without water, or the dilution volume was less than 50 µL (plasma: 0.6 M TCA: water = 1:2:1, v/v/v); the recoveries of PLP and PL were low; the recoveries of PLP were higher than 120% at the dilution volume of 100 µL; the recoveries of PL were within the normal range, and the PA chromatographic peak was deformed; and the recoveries were greater than 120%; considering the above results, extraction was performed according to plasma: 0.6 M TCA: ultrapure water = 1:2: Incubation times and temperatures [39][40][41] were investigated under established extraction conditions, as shown in Table S1. PLP, PA, and PL showed good recoveries under different incubation conditions; the longer the incubation time, the lower the recoveries of analytes; and the incubation condition of 50 • C for 5 min was selected considering the rapidity and efficiency of pretreatment.

Derived Condition Results
We studied the influence of semicarbazide concentration on the derivatization reaction and the optimum time and temperature of the derivatization reaction. The result is shown in Figure 2. The shorter the PLP reaction time, the higher the response, the higher the reaction temperature, and the faster the reaction tends to be stable; the lower the PL temperature, the lower the response, and there is a higher response value between 30 and 50 • C. For the purpose of rapid reaction, 50 • C was selected to react in the dark for 20 min. The mixed standard reacted completely with semicarbazide at a concentration ratio of 1:50, and PLP and PL in the sample could react completely with semicarbazide when the concentration of semicarbazide exceeded 50 times that of the analyte, while the derivatization reaction of some PA with semicarbazide could also be seen by the trend of PA. Incubation times and temperatures [39][40][41] were investigated under established extraction conditions, as shown in Table S1. PLP, PA, and PL showed good recoveries under different incubation conditions; the longer the incubation time, the lower the recoveries of analytes; and the incubation condition of 50 °C for 5 min was selected considering the rapidity and efficiency of pretreatment.

Derived Condition Results
We studied the influence of semicarbazide concentration on the derivatization reaction and the optimum time and temperature of the derivatization reaction. The result is shown in Figure 2. The shorter the PLP reaction time, the higher the response, the higher the reaction temperature, and the faster the reaction tends to be stable; the lower the PL temperature, the lower the response, and there is a higher response value between 30 and 50 °C. For the purpose of rapid reaction, 50 °C was selected to react in the dark for 20 min. The mixed standard reacted completely with semicarbazide at a concentration ratio of 1:50, and PLP and PL in the sample could react completely with semicarbazide when the concentration of semicarbazide exceeded 50 times that of the analyte, while the derivatization reaction of some PA with semicarbazide could also be seen by the trend of PA.

2D-LC System Online Processing Capability Results
The loading capacity of the 2D-LC column is up to 600 µL, while that of a conventional liquid chromatography column usually does not exceed 50 µL, so the separation capacity and sensitivity of the system can be improved. After pretreatment, 10, 50, 100, 200, 300, and 400 µL of the added samples at 10 µmol/L were injected; PLP, PA, and PL peak areas (As) were used as dependent variables, and injection volume (Am) was used as the independent variable (X) to establish a linear relationship between injection volume and peak areas; and the regression curve was fitted according to the least squares method to obtain the fitting equation Table 4. The results show that there is a good linear relationship between the injection volume and the peak area in the range of 10 to 400 µL, and R 2 is greater than 0.99. Large volume injection did not cause linear distortion of PLP, PA, and PL. The chromatographic peaks of PLP, PA, and PL were not distorted or shifted, showing that this two-dimensional liquid chromatography has good on-line enrichment ability for PLP, PA, and PL in samples, as shown in Figure 3.
capacity and sensitivity of the system can be improved. After pretreatment, 10, 50, 100, 200, 300, and 400 μL of the added samples at 10 μmol/L were injected; PLP, PA, and PL peak areas (As) were used as dependent variables, and injection volume (Am) was used as the independent variable (X) to establish a linear relationship between injection volume and peak areas; and the regression curve was fitted according to the least squares method to obtain the fitting equation Table 4. The results show that there is a good linear relationship between the injection volume and the peak area in the range of 10 to 400 μL, and R 2 is greater than 0.99. Large volume injection did not cause linear distortion of PLP, PA, and PL. The chromatographic peaks of PLP, PA, and PL were not distorted or shifted, showing that this two-dimensional liquid chromatography has good on-line enrichment ability for PLP, PA, and PL in samples, as shown in Figure 3.

2D-LC Transfer Capability Assay Results
The transfer capability of this system was assessed with transfer recovery (Rtr) and transfer precision (CV%) of samples at different concentrations, as shown in Table 5. The average transfer recoveries of PLP, PA, and PL at low, medium, and high concentrations were more than 90%, demonstrating complete transfer from the extraction column to the analytical column during column-to-column switching. The CV% of PLP, PA, and PL at different concentrations were within 8.9%, and the transfer stability of the 2D-LC system was good.

2D-LC Transfer Capability Assay Results
The transfer capability of this system was assessed with transfer recovery (Rtr) and transfer precision (CV%) of samples at different concentrations, as shown in Table 5. The average transfer recoveries of PLP, PA, and PL at low, medium, and high concentrations were more than 90%, demonstrating complete transfer from the extraction column to the analytical column during column-to-column switching. The CV% of PLP, PA, and PL at different concentrations were within 8.9%, and the transfer stability of the 2D-LC system was good.

Selectivity
PLP, PA, and PL were isolated under the liquid phase conditions described above. The retention times of PLP, PA, and PL were 5.1 min, 6.3 min, and 7.1 min, respectively. Representative chromatograms of blank plasma samples and spiked low concentration samples are shown in Figure 4. The retention time and peak profile of PLP, PA, and PL peaks did not change in the added samples. The method has good selectivity and is not influenced by endogenous substances in biological matrices. PLP, PA, and PL were isolated under the liquid phase conditions described above. The retention times of PLP, PA, and PL were 5.1 min, 6.3 min, and 7.1 min, respectively. Representative chromatograms of blank plasma samples and spiked low concentration samples are shown in Figure 4. The retention time and peak profile of PLP, PA, and PL peaks did not change in the added samples. The method has good selectivity and is not influenced by endogenous substances in biological matrices.

LOD, LOQ, and Linearity
The results of LOD and LOQ for PLP, PA, and PL are shown in Table 6. The LOD of PLP, PA, and PL was 0.1, 0.2, and 4 nmol/L, respectively, and the LOQ of PLP, PA, and PL was 0.2, 0.4, and 20 nmol/L, respectively, in the standard curve based on ultra-pure water. Using water as the matrix to make standard curves, PLP, PA, and PL had an excellent linear relationship within the quantitative range, R 2 > 0.99, repeated three times, and the RSD% was within 4%. The LOD and LOQ of the matrix-added samples were the same as those of the standard, which reached the same sensitivity as the existing HPLC-FLD and LC-MS methods [15,25,[42][43][44][45].
When generating the matrix standard curves, to reduce endogenous interference, the plasma matrix was diluted 50 times and 100 times with ultrapure water to explore suitable treatment methods. The test results are shown in Table S2. By comparing the relative standard deviations (RSDs) of plasma matrix curves and standard curves, plasma was diluted 50 and 100 times. Plasma diluted 50 times had less endogenous interference, and

LOD, LOQ, and Linearity
The results of LOD and LOQ for PLP, PA, and PL are shown in Table 6. The LOD of PLP, PA, and PL was 0.1, 0.2, and 4 nmol/L, respectively, and the LOQ of PLP, PA, and PL was 0.2, 0.4, and 20 nmol/L, respectively, in the standard curve based on ultra-pure water. Using water as the matrix to make standard curves, PLP, PA, and PL had an excellent linear relationship within the quantitative range, R 2 > 0.99, repeated three times, and the RSD% was within 4%. The LOD and LOQ of the matrix-added samples were the same as those of the standard, which reached the same sensitivity as the existing HPLC-FLD and LC-MS methods [15,25,[42][43][44][45]. When generating the matrix standard curves, to reduce endogenous interference, the plasma matrix was diluted 50 times and 100 times with ultrapure water to explore suitable treatment methods. The test results are shown in Table S2. By comparing the relative standard deviations (RSDs) of plasma matrix curves and standard curves, plasma was diluted 50 and 100 times. Plasma diluted 50 times had less endogenous interference, and the plasma matrix curve was closer to the standard curves (RSD% < 15%), with remarkable parallelism and the linear relationship (R 2 > 0.99) as the method verification object. The results proved that ultrapure water could not be used as an equivalent substitute for the plasma matrix, and water substitute matrix also led to high matrix effects and analytical inaccuracies in LC-MS/MS [43].

Precision, Accuracy and Recovery
Four levels with replicates (n = 5) were analyzed for intra-day and inter-day quality control assays. The results are shown in Table 7. The intra-day precision values were <12.6%. The accuracy varied from −7.61% to 15.38%. For the acceptance criteria for precision and accuracy, the results should conform to a coefficient of variation (CV) of ≤15% except for a coefficient of variation of LLOQ ≤ 20. The recovery values ranged from 90.00% to 113.90% with variability (CV) of <12.51%. The results showed that the method can be used for the quantitative analysis of PLP, PA, and PL in pig, mouse, and rat plasma. Table 7. Intra-and inter-day precision (CV), accuracy (bias), and recovery of PLP, PA, and PL in plasma of pigs, mouse and rats.

Stability
The stability of the method was expressed as relative standard deviation (RSD%). Table 8 shows the short-term stability results of pig, mouse, and rat plasma QCL, QCM and QCH samples after being placed at 4 • C for three days. The RSD% of pig plasma was 2.3% to 13.66%, mouse plasma was 2.6% to 13.37%, and rat plasma was 2.11% to 14.97%, suggested that the samples were placed at 4 • C for three days without analyte degradation, and the stability was good.

Method Application Results
A total of more than 30 valid samples were collected, and 2D-LC detection was performed after pre-processing. The results are shown in Table 9. Pig plasma PLP was 197~431 nmol/L, that of PL was 82~185 nmol/L, and that of PA was 3~15 nmol/L. Mouse plasma PLP was 131~256 nmol/L, that of PL was 25~130 nmol/L, and that of PA was 10~15 nmol/L. Rat plasma PLP was 151~300 nmol/L, that of PA was 9~51 nmol/L, and that of PL was 21~36 nmol/L. Table 9. Distribution of plasma PLP, PA, and PL in pigs, mice, and rats.

Discussion
For separating PLP, PA, and PL from endogenous substances, the stationary phase was selected for reversed-phase C18 and C8 column [29,[46][47][48][49][50][51], and the mobile phase was isocratic elution, which requires a suitable buffer added to adjust the pH and the elimination of overlapping peaks, tail peaks, and shoulder peaks. A Waters Summetry ® C18 column was selected as the 2D column, and the Aston TM ® SPY column (phenyl hexyl column, Hunan Demite instrument Co., Ltd., Changsha, China) was chosen as the 1D column, which had simultaneous retention of three analytes, and the mobile phases of the two dimensions had to be compatible. Acidic pH reduces the polarity of PLP and promotes its retention on the chromatographic column. Phosphate buffer and methanol were used as the original mobile phase. However, the pH of the phosphate buffer was unstable and easily crystallized when meeting methanol, and it blocked the internal pipeline, so the water phase was changed to a more stable ammonium acetate solution. In the 20 mM ammonium acetate solution with pH = 6.8, PLP, PA, and PL had the best resolution, but the PL peak was severely tailed. After adding acetonitrile and ammonium dihydrogen phosphate solution to the mobile phase, the PL peak tailing factor decreased, and the three compounds had good separation. The time running program is directly related to the transfer ability of the target from the extraction column to the analysis column and the sample load of the system; that is, the time window is too wide, the PLP peak loss is serious, the analyte sample load is small, the time window is too narrow, and the PL transfer is incomplete. Through the results of transfer recovery rates in different time windows, it was found that the target substance began to elute at 2.0 min and completely eluted at 3.5 min.
Vitamin B 6 binds tightly to proteins and must release the analyte from the protein under the premise of ensuring that the analyte in the sample is not degraded or metabolized. Precipitated protein method is a feasible sample preparation technique. Protein precipitants include trichloroacetic acid [52,53], perchloric acid [21,50,54], metaphosphoric acid [55], formic acid [56], methanol, acetonitrile, and so on. Among them, trichloroacetic acid and perchloric acid are most useful for releasing vitamin B 6 . Perchloric acid has strong acidity, which exceeds the acid-base tolerance range of the column. It often requires alkaline reagents to adjust the pH of the sample solution to protect the column, such as NaOH, KOH, and NH 4 OH, which were among many uncontrollable factors in the sample pretreatment. In contrast, trichloroacetic acid, methanol, and acetonitrile are ideal extraction reagents and were investigated. According to previous studies [39,40], the extraction reagent, dilution ratio, and incubation conditions of the extraction reagents were investigated. We found that, when plasma:0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and is incubated at 50 • C for 5 min, the extraction recovery rate was good.
The structural characteristics of PLP and PL determine that they do not have strong ultraviolet absorption and fluorescence. Through derivatization reactions, the analytes obtained or enhanced ultraviolet absorption and fluorescence characteristics, thereby improving the sensitivity and specificity. Zhang [57] investigated the effect of various chlorites, bisulfites, semicarbazide derivatization reagents, and methods on PLP derivatization. Sodium bisulfite precolumn derivatization obtained the highest signal-to-noise ratio but a short retention time. In contrast, the pre-column derivatization of semicarbazide produced a higher signal-to-noise ratio and a more acceptable on-column retention time. Considering the safety of derivatization reagents and the stability of derivatized products, the semicarbazide pre-column derivatization method is a more suitable derivatization method. In this study, we investigated the effects of the optimal time, temperature, and semicarbazide concentration on the semicarbazide-derivatization reaction. Semicarbazide requires more than a 50-fold concentration to fully derive with PLP and PL, and some PA is also reacted. The derivatization of the target reacts for 5 min at 50 • C in the dark, consistent with the extraction temperature, and TCA extraction and derivatization can be synchronized, followed by centrifugation to obtain the supernatant to reduce the depletion of the target during the extraction process.
HPLC detection methods have been relatively mature and easy to perform, with accurate determination and good repeatability. Fluorescence detection generally has good specificity and sensitivity. There are many studies of PLP, PA, and PL based on fluorescence detection, and a universal set of detection methods has been basically formed [29][30][31]. In this study, the LOQ of PLP was 0.2 nmol/L, that of PA was 0.4 nmol/L, and that of PL was 20 nmol/L. This result is similar to that obtained by other authors when quantifying vitamin B 6 samples, in which PLP is 3.5 to 4.5 nmol/L, PA is 1.2 to 6.3 nmol/L, and PL is 2.0 to 20 nmol/L [51,58]. Van der Ham M's quantitative limit for the determination of PLP in human cerebrospinal fluid by ultra-performance liquid chromatography-mass spectrometry is 0.38 nmol/L, as well as 0.09 nmol/L for PA and 1.54 nmol/L for PL [26]. It must be acknowledged that high performance liquid chromatography-mass spectrometry (HPLC/MS) methods have higher sensitivity than high performance liquid chromatography-ultraviolet and high-performance liquid chromatography-fluorescence detection (HPLC-FL) methods. The HPLC-MS/MS method for vitamin B 6 analysis has certain advantages, but HPLC-MS/MS detection equipment is expensive, it is difficult to operate the instrument, there are high maintenance costs, and it is not suitable for factory and laboratory routine application. Therefore, we have established a 2D liquid chromatography method for the simultaneous detection of PLP, PA, and PL in animal plasma, which provides more choices for the accurate quantification of PLP, PA, and PL. We also applied this method to animal plasma samples and determined the contents of PLP, PA, and PL in the plasma of pigs, mice, and rats. Due to the different ages of experimental pigs, the plasma PLP level of pigs has a large fluctuation range and is higher than the previous level. The plasma PLP and PL levels of mice are much higher than the reference values, which might be caused by not fasting them before sampling. The plasma PLP of rats is 151~300 nmol/L, that of PA is 9~51 nmol/L, and that of PL is 21~36 nmol/L, all of which are normal concentrations. This method has good applicability and accuracy.

Conclusions
In conclusion, we established for the first time a rapid, automatic, and low-cost 2D-LC method for simultaneous quantification of PLP, PA, and PL in animal plasma. The system provides a large-capacity injection with good detection sensitivity and strong antiinterference ability. After a short period of incubation at a high temperature, trichloroacetic acid releases plasma PLP, precipitates protein, and removes impurities in the pretreatment process. To improve the sensitivity of ultraviolet detection, PLP, PA, and PL were derivatized. The method has a low detection limit and LOQ and good specificity, precision, accuracy and short-term stability. This method has been successfully applied to plasma samples of pigs, mice, and rats, providing a new method for clinical trials and achieving the target of large-scale, low-cost, and wide-ranging sample detection.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani13081333/s1, Table S1: Recoveries of PLP, PA and PL extraction at different incubation times and temperatures; Table S2: Plasma matrix curves of pigs, mouse and rats.